Microtubule organization changes severely after mannitol and n-butanol treatments inducing microspore embryogenesis in bread wheat.

BACKGROUND: A mannitol stress treatment and a subsequent application of n-butanol, known as a microtubule-disrupting agent, enhance microspore embryogenesis (ME) induction and plant regeneration in bread wheat. To characterize changes in cortical (CMT) and endoplasmic (EMT) microtubules organization and dynamics, associated with ME induction treatments, immunocytochemistry studies complemented by confocal laser scanning microscopy (CLSM) were accomplished. This technique has allowed us to perform advanced 3- and 4D studies of MT architecture. The degree of MT fragmentation was examined by the relative fluorescence intensity quantification. RESULTS: In uni-nucleated mannitol-treated microspores, severe CMT and EMT fragmentation occurs, although a complex network of short EMT bundles protected the nucleus. Additional treatment with n-butanol resulted in further depolymerization of both CMT and EMT, simultaneously with the formation of MT aggregates in the perinuclear region. Some aggregates resembled a preprophase band. In addition, a portion of the microspores progressed to the first mitotic division during the treatments. Bi-nucleate pollen-like structures showed a high MT depolymerization after mannitol treatment and numerous EMT bundles around the vegetative and generative nuclei after n-butanol. Interestingly, bi-nucleate symmetric structures showed prominent stabilization of EMT. CONCLUSIONS: Fragmentation and stabilization of microtubules induced by mannitol- and n-butanol lead to new configurations essential for the induction of microspore embryogenesis in bread wheat. These results provide robust insight into MT dynamics during EM induction and open avenues to address newly targeted treatments to induce ME in recalcitrant species.

Analysis of wheat microspore embryogenesis induction by transcriptome and small RNA sequencing using the highly responsive cultivar "Svilena".

BACKGROUND: Microspore embryogenesis describes a stress-induced reprogramming of immature male plant gametophytes to develop into embryo-like structures, which can be regenerated into doubled haploid plants after whole genome reduplication. This mechanism is of high interest for both research as well as plant breeding. The objective of this study was to characterize transcriptional changes and regulatory relationships in early stages of cold stress-induced wheat microspore embryogenesis by transcriptome and small RNA sequencing using a highly responsive cultivar. RESULTS: Transcriptome and small RNA sequencing was performed in a staged time-course to analyze wheat microspore embryogenesis induction. The analyzed stages were freshly harvested, untreated uninucleate microspores and the two following stages from in vitro anther culture: directly after induction by cold-stress treatment and microspores undergoing the first nuclear divisions. A de novo transcriptome assembly resulted in 29,388 contigs distributing to 20,224 putative transcripts of which 9,305 are not covered by public wheat cDNAs. Differentially expressed transcripts and small RNAs were identified for the stage transitions highlighting various processes as well as specific genes to be involved in microspore embryogenesis induction. CONCLUSION: This study establishes a comprehensive functional genomics resource for wheat microspore embryogenesis induction and initial understanding of molecular mechanisms involved. A large set of putative transcripts presumably specific for microspore embryogenesis induction as well as contributing processes and specific genes were identified. The results allow for a first insight in regulatory roles of small RNAs in the reprogramming of microspores towards an embryogenic cell fate.

Carotenoids of aleurone, germ, and endosperm fractions of barley, corn and wheat differentially inhibit oxidative stress.

The antioxidant potential of carotenoids from aleurone, germ, and endosperm fractions of barley, corn, and wheat has been evaluated. HPLC analysis confirmed the presence of lutein and zeaxanthin carotenoids (nd-15139 µg/kg) in extracts of cereal grain fractions. The antioxidant properties using 2,2-diphenyl-1-picrylhydrazyl, oxygen radical absorbance capacity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays revealed significantly higher (P<0.001) antioxidant activity in the germ than in the aleurone and endosperm fractions. Using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, 2,2'azobis (2-amidinopropane)dihydrochloride (AAPH)-induced cell loss was effectively reduced by preincubating Caco-2, HT-29, and FHs 74 Int cells with carotenoid extracts. Moreover, carotenoid extracts reduced (P<0.001) AAPH-induced intracellular oxidation in the cell lines, suggesting antioxidant activity. Of the 84 antioxidant pathway genes included in microarray array analysis (HT-29 cells), the expressions of 28 genes were enhanced (P<0.05). Our findings suggest that carotenoids of germ, aleurone, and endosperm fractions improved antioxidant capacity and thus have the potential to mitigate oxidative stress.

Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development.

Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation.

Cell-free protein synthesis as a promising expression system for recombinant proteins.

Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay.

Chapter 2. Development of key technologies for high-throughput cell-free protein production with the extract from wheat embryos.

The cell-free translation system from wheat embryos had been considered to be inefficient as compared with the E. coli cell-based and cell-free protein production methods. However, it was revealed that the extract from extensively washed wheat embryo particles can provide a very productive cell-free protein synthesis system. Since then, the method has been improved, so that it fits the postgenomic researches. New mRNA configurations enabled us to synthesize many different proteins in parallel and to prepare large amounts of proteins, which fits the need for screening of suitable proteins for structural and functional analyses before large-scale production. The new reaction formats promoted the developments of new machines that perform highly parallel and highly productive protein synthesis reactions automatically. It was revealed that, by parallel synthesis of many proteins, much more multidomain proteins are produced in soluble forms in the wheat system than in the prokaryotic systems. The wheat system provides a rapid and cost-effective method for stable isotope labeling of proteins for NMR analyses. Selenomethionine substitution of proteins for X-ray crystallography through the cell-free synthesis was also achieved. Synthesis of some families of proteins that were difficult to be produced by conventional methods has been tested. At least, cytotoxic restriction enzymes were readily produced in a large amount. Some multisubunit proteins and cofactor-binding proteins could be synthesized by the method and were characterized successfully. Membrane proteins have also been tested, and a transporter was synthesized in an active form. Although some issues remains to be solved, we expect that the wheat cell-free protein synthesis system can contribute to the structural and functional genomics and to the future understanding of life.

Effects of aqueous eucalyptus extracts on seed germination, seedling growth and activities of peroxidase and polyphenoloxidase in three wheat cultivar seedlings (Triticum aestivum L.).

Evaluation of allelopathic effects of this plant on other near cultivations especially wheat is the aim of this study. Effects of water extracts of eucalyptus leaves examined on germination and growth of three wheat cultivar seeds and seedlings. Results showed that: germination percentage strongly decreased, leaf and root lengths also affected and dry and wet weights of both roots and shoots showed similar change patterns. Activities of peroxidase and polyphenoloxidase as antioxidant enzymes in roots and shoots measured. Activity of peroxidases increased in stress conditions and roots showed more increased enzyme activity than leaves. Activity of polyphenoloxidases increased only in one of three cultivars and again roots showed more activity of this enzyme in response to eucalyptus extract. Suggest that detoxification process were conducted mainly in roots of seedlings.

A novel way to express proline-selectively labeled proteins with a wheat germ cell-free protein synthesis system.

For high-throughput protein structural analyses, it is essential to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones involving Escherichia coli cells, have been developed, the number of overexpressed proteins exhibiting the same biological activities as those of the native ones is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the synthesized proteins that should function in solution were found to be in soluble forms. This suggests the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we developed a selective labeling technique for amino acids having amide functional groups (other than proline residues) involving the use of several inhibitors for transaminases. This paper in turn describes a proline-selective labeling technique. Based on our results, we have succeeded in constructing a complete amino acid selective labeling technique for the wheat germ cell-free protein synthesis system.

Oligonucleotide-directed gene repair in wheat using a transient plasmid gene repair assay system.

Oligonucleotide-directed gene repair is a potential technique for agricultural trait modification in economically important crops. However, large variation in the repair frequencies among the scientific reports indicates that there are many factors influencing the repair process. We report here a transient assay system using GFP as a reporter for testing the efficiency of plasmid DNA repair in cultured wheat cells. This assay showed that osmotic medium supplemented with 2,4-D increased the oligo-targeting frequency, and that the repair of a point mutation was more efficient than repair of a single base deletion mutation in cultured scutellum cells of immature wheat embryos. This study provides the first evidence that oligonucleotide-directed mutagenesis is applicable to regenerable cultured wheat scutellum cells.

Biochemical dissection of RNA silencing in plants.

Although RNA silencing was first discovered in plants, thus far it has been studied biochemically only in animals, where it is known as RNA interference (RNAi). In animals, two components of the RNAi pathway have been identified: Dicer, a multidomain RNase III that converts long double-stranded RNA (dsRNA) into small interfering RNA (siRNA) and the RNA-induced silencing complex (RISC), as siRNA-containing protein-RNA complex that targets complementary mRNA for destruction. We have developed methods for the biochemical dissection of plant RNA silencing. In this chapter, we describe in detail how to use wheat germ extract to study two distinct Dicer-like activities, RNA-dependent RNA polymerase (RdRP), and endogenous microRNA-programmed RISC activities. These comprehensive protocols should prove useful in the further dissection of the plant RNA silencing pathway, as well as for the validation of the predicted targets of endogenous plant microRNAs.

Effect of zearalenone treatment on the production of wheat haploids via the maize pollination system.

The ability of zearalenone (ZEN) to stimulate the growth of haploid wheat embryos formed following the pollination of wheat spikes with maize pollen was tested. The maize pollination system was used as a model to compare the activity of ZEN with that of auxin analogues. Three solutions, each with a different concentration of ZEN (6.0, 3.0 or 1.5 microM), and a solution of 2,4-dichlorophenoxyacetic acid (control) were tested for their effect on ovary swelling, frequency of embryo formation and the ability to regenerate plants. In total, 3,105 florets of 282 spikes from five different cultivars of hexaploid winter wheat ( Triticum aestivum L.) were pollinated with maize ( Zea mays L. cv. Gama) pollen and treated with the ZEN solutions. The highest concentration of ZEN (6.0 microM) was the most effective in inducing ovary swelling (84 swollen ovaries/100 pollinated florets) and increasing the frequency of embryo induction (18.9 embryos/100 pollinated florets), but these embryos were severely deformed. They had low capability to germinate in vitro, while callus was easily formed and indirect regeneration of plants was possible. The lowest ZEN concentration (1.5 micrroM) induced ovary swelling in 42.8/100 pollinated florets and embryo growth in 3.3 out of 100 pollinated florets. The embryos were regular in shape, and almost half of them germinated in vitro while callus induction from them failed. The concentration of 3 microM ZEN had an intermediate effect. The type of response of the various wheat genotypes was similar, while frequencies were different, with cv. Izolda being the most responsive. The results show that ZEN has some of the properties of an auxin analogue, while other effects of its action are unique. It can be used in the maize pollination system of doubled haploid production to replace auxin analogues when indirect regeneration of plants via callus tissue is planned.

Induced abnormality in Mir- and Earth grown Super Dwarf wheat.

Super-dwarf wheat grown on the Mir space station using the Svet "Greenhouse" exhibited morphological, metabolic and reproductive abnormalities compared with Earth-grown wheat. Of prominent importance were the abnormalities associated with reproductive ontogeny and the total absence of seed formation on Mir. Changes in the apical meristem associated with transition from the vegetative phase to floral initiation and development of the reproductive spike were all typical of 'Super-Dwarf' wheat up to the point of anthesis. Observation of ruptured anthers from the Mir-grown plants revealed what appeared to be normally developed pollen. These pollen gains, however, contained only one nuclei, while normal viable pollen is tri-nucleate. A potentially important difference in the flight experiment, compared with ground reference studies, was the presence of a high level of atmospheric ethylene (1,200 ppb). Ground studies conducted by exposing 'Super-Dwarf' wheat to ethylene just prior to anthesis resulted in manifestation of the same abnormalities observed in the space flight samples.

A biochemical framework for RNA silencing in plants.

RNA silencing phenomena were first discovered in plants, yet only the RNA interference pathway in animals has been subject to biochemical analysis. Here, we extend biochemical analysis to plant RNA silencing. We find that standard wheat germ extract contains Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs, as well as an RNA-dependent RNA polymerase activity that can convert exogenous single-stranded RNA into dsRNA. In this plant embryo extract, an endogenous microRNA (miRNA) that lacks perfect complementarity to its RNA targets nonetheless acts as a small interfering RNA. The miRNA guides an endonuclease to cleave efficiently wild-type Arabidopsis PHAVOLUTA mRNA, but not a dominant mutant previously shown to perturb leaf development. This finding supports the view that plant miRNAs direct RNAi and that miRNA-specified mRNA destruction is important for proper plant development. Thus, endonuclease complexes guided by small RNAs are a common feature of RNA silencing in both animals and plants.

Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase activation in Jurkat T-cell leukemia tumor cells.

The fermented extract of wheat germ, trade name Avemar, is a complex mixture of biologically active molecules with potent anti-metastatic activities in various human malignancies. Here we report the effect of Avemar on Jurkat leukemia cell viability, proliferation, cell cycle distribution, apoptosis, and the activity of key glycolytic/pentose cycle enzymes that control carbon flow for nucleic acid synthesis. The cytotoxic IC(50) concentration of Avemar for Jurkat tumor cells is 0.2 mg/ml, and increasing doses of the crude powder inhibit Jurkat cell proliferation in a dose-dependent fashion. At concentrations higher than 0.2 mg/ml, Avemar inhibits cell growth by more than 50% (72 h of incubation), which is preceded by the appearance of a sub-G(1) peak on flow histograms at 48 h. Laser scanning cytometry of propidium iodide- and annexin V-stained cells indicated that the growth-inhibiting effect of Avemar was consistent with a strong induction of apoptosis. Inhibition by benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone of apoptosis but increased proteolysis of poly(ADP-ribose) indicate caspases mediate the cellular effects of Avemar. Activities of glucose-6-phosphate dehydrogenase and transketolase were inhibited in a dose-dependent fashion, which correlated with decreased (13)C incorporation and pentose cycle substrate flow into RNA ribose. This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. Avemar exhibits about a 50-fold higher IC(50) (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.

[Wheat androgenic embryoids and calli: data of scanning electron microscopy]. / Androgennye émbrioidy i kallusy pshenitsy: dannye skaniruiushchei élektronnoi mikroskopii.

The surface of wheat androgenic embryoids and calli at different developmental stages was studied using SEM. The embryoids were already characterized by regular cell divisions at the early developmental stages, while the calli were represented by irregular cell conglomerates. This trend was preserved during further development of androgenic structures. SEM studies of the surface of so-called secondary embryoids confirmed these observations.

Cytodifferentiation and transformation of embryogenic callus lines derived from anther culture of wheat.

Three types of callus tissues established from anther culture of eleven doubled haploid (DH) lines of wheat (Triticum aestivum L.) were evaluated for their ability in enhancing friable embryogenic (Type II) culture differentiation and genetic transformation. Differences between types of callus inocula were highly significant (P < 0.001), suggesting that the quality of the initial callus explant is of profound importance in encouraging the proliferation of Type II cultures. Other factors found to be crucial included weekly subculture of friable embryogenic callus tissues on a maintenance medium containing 30 microM dicamba and a predominance of amino-acid nitrogen supplement. Transfer and integration of the beta-glucuronidase gene was also affected by the type of inoculum when suitable embryogenic cell cultures were transformed using silicon carbide whiskers and high velocity microprojectiles. Expression of the hygromycin phosphotransferase selectable marker gene sequence was confirmed in all the stably transformed cell lines maintained on selection media containing lethal levels of hygromycin. Comparatively, there were differences in the frequency of regenerable, transgenic clonal segments between whisker-treated and microprojectile bombarded tissues mainly as a result of the fact that cultures vortexed with whiskers were more capable of post-treatment cell proliferation and embryo differentiation than those bombarded with cDNA-coated microprojectiles. Conditions for obtaining these results are outlined and discussed in relation to the suitability of the two transformation strategies for producing transgenic cell aggregates of wheat.

Changes in abundance of an abscisic acid-responsive, early cysteine-labeled metallothionein transcript during pollen embryogenesis in bread wheat (Triticum aestivum).

A clone for an embryoid-abundant, early cysteine-labeled metallothionein (EcMt) gene has been isolated from a wheat pollen embryoid cDNA library. The transcript of this gene was only expressed in embryogenic microspores, pollen embryoids, and developing zygotic embryos of wheat. Accumulation of the EcMt mRNA showed a direct and positive correlation with an increase of the plant hormone, abscisic acid (ABA) in developing pollen embryoids. Treating cultures with an inhibitor of ABA biosynthesis, fluridone, suppressed not only ABA accumulation but also the appearance of the EcMt gene transcript and the ability of microspores to form embryoids. These results suggest that the EcMt gene may act as a molecular marker for pollen embryogenesis because ABA biosynthesis is accompanied by the increased expression of the EcMt transcript that coincides with the differentiation of pollen embryoids in wheat anther cultures.

Uptake and transient expression of chimeric genes in seed-derived embryos.

Uptake of DNA in dry and viable embryos of wheat by imbibition in DNA solution was detected by monitoring the transient expression of chimeric genes. Gene expression vectors used in this study contained a neomycin phosphotransferase (NPT) II reporter gene fused to various promoters. Some of the chimeric "neo" genes were shown to yield reproducibly NPT II activity in germinating embryos. This NPT II activity was increased markedly when the neo genes were carried by a vector capable of autonomous replication. Dimers of wheat dwarf virus, a monopartite gemini virus, were thus shown to be effective in amplifying the transient expressed NPT II activity in embryos of several cereals. These and other observations indicate that the observed transient expression really results from DNA uptake and expression in plant embryo cells and is not due to contaminating microorganisms.

The nucleotide sequence of a cDNA clone encoding the wheat Em protein.

The nucleotide sequence of a cDNA clone isolated from developing wheat embryos and encoding the Em protein is reported. The entire coding region for Em and the 3' non-translated flank are contained within this clone. The amino acid sequence deduced for Em is very rich in glycine (18 mol%) as well as both basic and acidic residues. The molecular weight of the protein is ca. 9,900 daltons. The deduced sequence is supported by direct amino acid sequencing of cyanogen bromide cleavage fragments obtained from purified Em protein. Em is shown by Southern blots to be a product of a gene family of approximately ten members.